Legionella pneumophila (Lp) is the agent of Legionnaires' disease pneumonia. In its aquatic habitat, Lp survives as an intracellular parasite of protozoans, and after inoculation into the lung, it flourishes within alveolar macrophages. Previously, we discovered an Lp gene (pilD) whose product is homologous with PilD, which, in other Gram-negatives, mediates pilus biogenesis and type II protein secretion. Indeed, an Lp pilD mutant lacked pili. More importantly, the pilD mutant, but not a pilin mutant, was defective for infection of amoebae and macrophages, suggesting that Lp has a type II secretion system that promotes infection. During this last grant period, we confirmed Lp has a type II system (lsp) that mediates secretion of many proteins, including novel enzymes, and is critical for infection of both protozoa and human cells. Some of our other data suggested that the Lp peptidyl-prolyl isomerase Mip and SurA have a role in the secretion process. Presently, Lp is the only known system for studying type II secretion in an intracellular pathogen. In the last grant period, we also demonstrated that lsp mutants are greatly impaired for survival in the lungs of A/J mice, with the severity of their defect indicating that Lp type II secretion is a key virulence determinant that may be involved in more than just macrophage infection. Finally, we made the novel observation that lsp is required for growth at 12-25 degrees C. Thus, Lp type II secretion is uniquely critical for intracellular infection, virulence, and low-temperature growth. In the current proposal, we aim to identify the type II exoproteins that are critical for intracellular infection, confirm the role of Mip and SurA in secretion, and determine how type II secretion promotes in vivo survival as well as low-temperature growth in water and amoebae. The results of these studies will i) increase our understanding of Lp physiology and pathogenesis, ii) provide new insight into bacterial protein secretion, intracellular infection, and low-temperature growth, and iii) have implications for other important human pathogens, including both other intracellular parasites and extracellular pathogens, such as Vibrio, Pseudomonas, and Burkholderia that survive at low-temperature and have type II systems.